Epigenetic Regulation of Cell and Tissue Aging

正常的人类细胞对潜在的致癌事件做出反应，如端粒缩短、DNA损伤和 通过不可逆地抑制生长激活癌基因，这种生长具有一种称为细胞的特征表型 衰老。这一过程最早是在细胞培养中发现的，最近在体内被证实为一种关键的 抑制人类肿瘤恶性进展的机制。Rb/p16INK4a，但不是p53， 调节培养中的人黑素细胞和体内黑素细胞痣的衰老途径。这 衰老反应可能是由于染色质的修饰，因为Rb络合物来自衰老 黑素细胞含有更高水平的组蛋白脱乙酰酶活性和拴系的HDAC1。我们发现了 中等水平的HDAC1的表达驱动着染色质的顺序和协同活动 重塑效应器，包括布拉玛在RB/HDAC1百万复合体中的瞬时招募，稳定 Rb与染色质的结合、yH_2AX DNA损伤灶的形成和整体异染色化。 这些染色质的变化与典型的衰老标志物的表达相吻合。基于这些 我们假设基因组维持的表观遗传变化会导致细胞和生物的 衰老。具体来说，我们建议： 目标1：a)确定Brm1相关的染色质重塑活动是否需要 衰老相关异染色灶(SAHF)的组装和Rb诱导的细胞衰老 人类黑素细胞。这一假设将在原代培养中使用病毒表达系统进行验证， 基于功能细胞的检测，包括HDAC1诱导系统和基于染色质的检测。B)定义 Brm1/Rb络合物组成的衰老依赖性变化。我们将使用基于高效液相的SIZE 络合物的排除分级、免疫分离的高效液相色谱分离、GST-Pull-Down和MS 光谱分析。C)确定肝脏中是否存在染色质重塑复合体的“保守核心” 来自古老的动物。 目的2：确定衰老之前的表观遗传变化与DNA之间的分子联系 损坏反应。我们将确定H_2AX的磷酸化和/或特异性的甲基化 组蛋白H3残基表明染色质重塑引发的“DNA反应”。 总之，这项提案中包括的研究旨在解开顺序和合作的作用 组蛋白修饰蛋白在细胞衰老和组织衰老中的作用。如果成功，我们的研究将 帮助定义“细胞衰老的组蛋白密码”；从而为干预开辟了新的途径 以人类表观基因组为目标。

Normal human cells respond to potentially oncogenic events such as short telomeres, DNA damage and activating oncogenes by irreversibly arresting growth with a characteristic phenotype termed cellular senescence. This process, first identified in cell culture, has been recently confirmed in vivo as a critical mechanism that curtails the malignant progression of human tumors. The RB/p16INK4a, but not p53, pathway regulates the senescence of human melanocytes in culture and melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase activity and tethered HDAC1. We have found that expression of moderate levels of HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma into RB/HDAC1 mega complexes, stable association of RB with chromatin, formation of yH2AX DNA damage foci and global heterochromatization. These chromatin changes coincided with expression of typical markers of senescence. Based on these findings we hypothesize that epigenetic changes in genome maintenance lead to cellular and organismal aging. Specifically, we propose to: Aim 1: A) To determine whether the Brm1-associated chromatin remodeling activity is required for the assembly of senescent-associated heterochromatic foci (SAHF) and RB-induced cellular senescence in human melanocytes. This hypothesis will be tested using viral expression systems in primary cultures, functional cell-based assays including an HDAC1-inducible system and chromatin-based assays. B) Define senescent-dependent changes in the composition of Brm1/RB complexes. We will use HPLC-based, size exclusion fractionation, immuno-isolation of complexes by HPLC fractionation, GST-pull-downs and Mass Spectrometry. C) Determine whether a "conserved core" of chromatin remodeling complexes exists in livers from old animals. Aim 2: To define molecular links between epigenetic changes that precede senescence and the DNA damage response. We will determine whether phosphorylation of H2AX and/or methylation of specific Histone H3 residues are indicative of a "DNA response" triggered by chromatin remodeling. Together, the studies included in this proposal are aimed at unraveling the role of sequential and cooperative functions of histone modifying proteins in cellular senescence and tissue aging. If successful, our studies will help define a "histone code for cellular aging"; and consequently open new avenues for interventions that target the human epigenome.